Name: Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells (Video) | JoVE Journal
Uploaded: 2008-06-17T17:21:00.000+00:00
Duration: 12 min 42 s
Description: Learn to analyze mitochondrial respiratory chain complexes in human embryonic stem cells using in gel activity assays. This method enables assessment of mitochondrial function for stem cell biology research.

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5 minutes

Human embryonic stem cell studies in the Teitell lab are currently supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank Dr. Carla Koehler (UCLA) and members of the Koehler laboratory for providing advice and insights in these and additional studies.

High Resolution Clear Native Gel Electophoresis (hrCNE) for Mitochondrial Complex In-Gel Activity Assays
Human embryonic stem cells (hESCs) were maintained on a monolayer of &#x02E0;-irradiated or mitomycin C-treated mouse embryonic fibroblasts (MEFs), then switched to growth on Matrigel for 5 days in MEF-conditioned medium using standard conditions. To remove contaminating MEFs, hESCs were re-plated on Matrigel and grown in MEF-conditioned medium for an additional 3 days. Immunofluorescence microscopy for pluripotency markers Oct-4 and SSEA-4 was used to confirm the lack of hESC differentiation.
<ol>
<li>Wash cells with warm 1x PBS, pH 7.4, followed by trypsinization (1ml of 1x trypsin per well in a 6-well plate) for 5 min. </li>
<li>Add an equal volume of fresh Trypsin Inhibitor (1mg/ml in 1x PBS, pH 7.4). Harvest cells and centrifuge at 200g, 5 min, room temperature.</li>
<li>Discard the supernatant and resuspend pelleted cells in 0.8 - 1.7ml (based upon the size of the cell pellet) of ice cold mitochondria isolation buffer (83mM sucrose; 6.6mM imidazole/HCl, pH 7.0) with freshly prepared 1&acute; Protease Inhibitor Cocktail. Incubate on ice for 10 min.</li>
<li>Dounce cells with a homogenizer (~40 - 50 strokes). Stain an aliquot of the dounced cells with trypan blue to check for completeness of homogenization using a light microscope. </li>
<li>Centrifuge at 20,000g for 10 min at 4&deg;C to obtain a crude mitochondrial pellet that also contains nuclei and larger cell fragments. Decant and discard the supernatant and weigh the precipitated crude mitochondrial pellet. The pellet can be stored at -80 &deg;C following instant freezing in liquid nitrogen if this is a stopping point. NOTE: Differential centrifugation or other gradient-based procedures can be used for isolating and collecting a pure mitochondrial membrane fraction. See ref. 4 for details. This alternative approach will yield the mitochondrial complex activity per mg of mitochondrial protein instead of complex activity per mg of cell weight or cell number. The concentration of mitochondrial proteins will need to be determined after purification to make this comparison. Also, a different isolation buffer may be required for this approach. For solubilization of the pure mitochondrial membrane fraction, see the note following step 6 below. </li>
<li>Solubilize the crude mitochondrial pellet on ice by vortexing with ice cold solubilization buffer (50mM NaCl, 50mM imidazole/HCl pH 7.0, 1mM EDTA, 2mM 6-aminohexanoic acid). 6-aminohexanoic acid can be replaced with the 1x Protease Inhibitor Cocktail. Use 35&micro;l of solubilization buffer per 20mg of pellet weight. Add 20&micro;l of 10% w/v digitonin per 20mg of pellet weight and mix by flicking the tube. Incubate for 5-10 min on ice. Dodecyl-b-D-maltoside or triton X-100 can be substituted for digitonin to solubilize the proteins. The choice of detergent and its quantity may affect the formation of supercomplexes or multimeric forms of mitochondrial complexes. For more information, see ref.1. NOTE: The quantity of detergent required to solubilize a pure mitochondrial membrane fraction corresponds to a detergent/protein weight ratio ranging from 2-6g/g. The optimal ratio to solubilize mitochondrial membranes is 2.5g/g for dodecyl-b-D-maltoside, 3g/g for triton X-100, and 6g/g for digitonin. </li>
<li>Centrifuge the solubilized pellet at 100,000g for 15 min at 4&deg;C. Collect the clear supernatant.</li>
<li>Add loading buffer to a final 5% w/v glycerol and 0.01% w/v Ponceau S (use a 50% w/v glycerol, 0.1% w/v Ponceau S loading buffer stock solution).</li>
<li>Load gel wells with a volume that contains 20-40mg of the crude mitochondrial pellet per lane. Use an acrylamide gradient gel (4 -13%) with a 3.5% stacking gel. NOTE: Gel preparation and electrophoresis conditions: For gel preparation, use an acrylamide/bis-acrylamide (AA/bisAA) 3 mix as follows: 48g of acrylamide and 1.5g of bisacrylamide (49.5% T, 3% C. Where T is total concentration of AA and bisAA and C is the percentage of crosslinker (bisAA) to total monomer). Gel buffer (3&acute;): 1.5M 6-aminohexanoic acid; 75mM imidazole, pH 7.0</li>
</ol>

<table style="padding-left: 60px;" border="0">
<tbody>
<tr>
<td>Gradient gel:</td>
<td>4%</td>
<td>13%</td>
<td>Stacking gel:</td>
<td>3.5%</td>
</tr>
<tr>
<td>AA/bisAA 3 mix</td>
<td>2 ml</td>
<td>6.5 ml</td>
<td>&nbsp;</td>
<td>0.6 ml</td>
</tr>
<tr>
<td>Gel buffer 3&acute;</td>
<td>8.3 ml</td>
<td>8.3 ml</td>
<td>&nbsp;</td>
<td>2.7 ml</td>
</tr>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>5 g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>H2O</td>
<td>14.7 ml</td>
<td>5 ml</td>
<td>&nbsp;</td>
<td>4.7 ml</td>
</tr>
<tr>
<td>Total volume</td>
<td>25 ml</td>
<td>25 ml</td>
<td>&nbsp;</td>
<td>8 ml</td>
</tr>
<tr>
<td>10% APS</td>
<td>139 &micro;l</td>
<td>125 &micro;l</td>
<td>&nbsp;</td>
<td>67 &micro;l</td>
</tr>
<tr>
<td>TEMED</td>
<td>14 &micro;l</td>
<td>12.5 &micro;l</td>
<td>&nbsp;</td>
<td>6.7 &micro;l</td>
</tr>
</tbody>
</table>

Anode buffer: 25mM imidazole/HCl, pH 7.0 Cathode buffer: 50mM Tricine, 7.5mM imidazole/HCl, 0.02% w/v dodecyl-b-D-maltoside, 0.05% w/v deoxycholate, pH 7.0 Pour and run gels in a cold room (4-7 &deg;C). The initial voltage used is 100V. When the sample has entered the stacking gel, the voltage is raised to 500V with the current limited to 15mA (for 0.15 &acute; 14 &acute; 14-cm gels). Electrophoresis is stopped after 3-4 h when the sharp line of the red Ponceau S dye approaches the gel front. Alternatively, a gel can be run overnight with constant voltage at 70-100V.
<ol>
<li>In-gel activity assays. The time of incubation for each assay depends on the efficiency of protein solubilization and the amount of material loaded per well. Complexes I, IV and V usually yield stronger signals than complexes II and III.</li>
</ol>
Complex I (CI)
Incubate the gel slice with CI buffer (5mM Tris-HCl, pH 7.4; 0.1mg/ml NADH; 2.5mg/ml NBT) at room temperature for several hours.
CI buffer: Add per 100ml of 5mM Tris-HCl, pH 7.4:
0.01g NADH
0.25g NBT
Fix the gel in 50% methanol and 10% acetic acid for 15-45 min. The fixed gel is preserved in 10% acetic acid.
Complex II (CII)
Incubate the gel slice with CII buffer (20mM sodium succinate; 0.2mM phenazine methasulfate; 2.5mg/ml NBT; 5mM Tris-HCl pH 7.4) at room temperature for several hours.
CII buffer: Add per 100ml of 5mM Tris-HCl, pH 7.4:
200&micro;l 1M sodium succinate
80&micro;l phenazine methasulfate (250mM PMS dissolved in DMSO)
0.25g NBT
Fix the gel as described for Complex I
Complex III (CIII)
Incubate the gel slice with CIII buffer (50mM sodium phosphate, pH 7.2; 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB)) at room temperature for several hours.
CIII buffer: Add per 100ml of 50mM sodium-phosphate, pH 7.2:
50mg diaminobenzidine tetrahydrochloride
Fix the gel as described for Complex I
Complex IV (CIV)
Incubate the gel slice with CIV buffer (50mM sodium phosphate, pH 7.2; 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB), 50&micro;M horse heart cytochrome c) at room temperature for several hours.
CIV buffer: Add per 100ml of 50mM sodium-phosphate, pH 7.2:
50mg diaminobenzidine tetrahydrochloride
0.1g horse heart cytochrome c
Fix the gel as described for Complex I
Complex V (CV)
Incubate the gel slice with CV buffer (35mM Tris-base, 270mM glycine, pH 8.3 at 25&deg;C, 14mM MgSO4, 0.2% w/v Pb(NO3)2, 8mM ATP) at room temperature for several hours.
CV buffer: Per 100ml of solution add:

<table border="0">
<tbody>
<tr>
<td>Tris-base</td>
<td>0.424g</td>
</tr>
<tr>
<td>Glycine</td>
<td>2.026g</td>
</tr>
<tr>
<td>MgSO4</td>
<td>0.345g</td>
</tr>
<tr>
<td>Pb(NO3)2</td>
<td>0.29g</td>
</tr>
</tbody>
</table>

No need to adjust pH (should be 8.3) - do not use acid or base. Filter and add

<table border="0">
<tbody>
<tr>
<td>ATP</td>
<td>0.441g</td>
</tr>
</tbody>
</table>

Fix the gel in 50% methanol for 30 min and transfer to water.

<ol><li>Wittig, I., Braun, H. -P., Sch˫gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blue+native+PAGE%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Blue native PAGE.</a> Nature Protocols. 1, 418-428 (2006).</li>
<li>Wittig, I., Karas, M., Sch˫gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+Resolution+Clear+Native+Electrophoresis+for+In-gel+Functional+Assays+and+Fluorescence+Studies+of+Membrane+Protein+Complexes.++6%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Proteomics%22%5BJournal%5D)">High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes. 6.</a> Molecular & Cellular Proteomics. 6, 1215-1225 (2007).</li>
<li>Wittig, I., Carrozzo, R., Santorelli, F. M., Sch˫gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+assays+in+high-resolution+clear+native+gels+to+quantify+mitochondrial+complexes+in+human+biopsies+and+cell+lines%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.</a> Electrophoresis. 28, 3811-3820 (2007).</li>
<li>Castle, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+Organelles+from+Mammalian+Cells%5BTitle%5D)+AND+%22Current+Protocols+in+Protein+Science%22%5BJournal%5D)">Purification of Organelles from Mammalian Cells.</a> Current Protocols in Protein Science. Coligan, J. E., Dunn, B. M., Speicher, D. W., Wingfield, P. T. 4.2, John Wiley & Sons, Inc. New York. 4.2.1-4.2.57 (2007).</li>
</ol>

In this video, we have described the extraction of mitochondrial protein complexes from human embryonic stem cells, their separation by high resolution clear native gel electrophoresis (hrCNE), and the subsequent analysis by in-gel catalytic activity assays. hrCNE resolves mitochondrial membrane protein complexes at a resolution comparable to that of blue native gel electrophoresis and is superior for in-gel activity assays. This technique can be employed not only to quantify mitochondrial respiratory complexes I-V but a...

<table><tbody><tr><td>0.5M EDTA</td><td>Fisher Scientific</td><td>BP120-1</td><td>adjust pH to 8.0 with NaOH </td></tr><tr><td>1M NaCl</td><td>Sigma-Aldrich</td><td>S3014-500MG</td><td></td></tr><tr><td>1M Sodium succinate</td><td>Fisher Scientific</td><td>S413-500</td><td></td></tr><tr><td>0.1M Sodium phosphate buffer pH 7.2</td><td>Fisher Scientific</td><td>BP332-1 and BP329-1</td><td>Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C </td></tr><tr><td>1M Tricine, pH 7.0 (adjusted with NaOH)</td><td>EMD Millipore</td><td>9010</td><td>store at 4 C </td></tr><tr><td>1M imidazole/HCl, pH 7.0</td><td>Sigma-Aldrich</td><td>I-2399</td><td>store at 4 C</td></tr><tr><td>1M Tris-HCl, pH 7.4</td><td>Fisher Scientific</td><td>BP153-1</td><td></td></tr><tr><td>20% (wt/vol) dodecyl-b-D-maltoside (DDM)</td><td>Fluka</td><td>44205-500MG</td><td>Dissolved in water. Store 1 ml aliquots at &amp;#8211;20 C.</td></tr><tr><td>10% (wt/vol) digitonin (&amp;gt;50% purity, used without recrystallization)</td><td>Fluka</td><td>37008-1G</td><td>Dissolved in water. Store 0.1&amp;#8211;1 ml aliquots at &amp;#8211;20 C. Should be warmed up to 95 C before use.</td></tr><tr><td>2M 6-aminohexanoic acid</td><td>Fluka</td><td>07260-100G</td><td>store at 4 C</td></tr><tr><td>10x Ponceau S/glycerol stock solution</td><td>Sigma-Aldrich</td><td>P-3504 and G6279-1L</td><td>0.1% Ponceau S, 50% glycerol, (wt/vol) in water.</td></tr><tr><td>10% w/v sodium deoxycholate</td><td>Fluka</td><td>30970-25G</td><td>Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature.</td></tr><tr><td>250 mM phenazine methasulfate (PMS)</td><td>TCI America</td><td>P0083</td><td>Dissolved in DMSO and aliquoted. Store at &amp;#8211;20 C.</td></tr><tr><td>Nitro-blue tetrazolium (NBT)</td><td>Amresco</td><td>0329-1G</td><td></td></tr><tr><td>3,3&amp;rsquo;-diaminobenzidine tetrahydrochloride (DAB)</td><td>TCI America</td><td>D0078</td><td></td></tr><tr><td>Pb(NO3)2</td><td>Fisher Scientific</td><td>L62-100</td><td></td></tr><tr><td>MgSO4</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Tris base </td><td>Fisher Scientific</td><td>BP152-10</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>G45-212</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo;-triphosphate disodium salt (ATP)</td><td>Sigma-Aldrich</td><td>A2383-5G</td><td>Grade I, minimum 99%</td></tr><tr><td>beta-nicotineamide adenine dinucleotide (NADH) reduced form </td><td>Sigma-Aldrich</td><td>N-8129</td><td></td></tr><tr><td>Cytochrome c From horse heart</td><td>Sigma-Aldrich</td><td>C2506-250MG</td><td></td></tr><tr><td>Sucrose </td><td>Fisher Scientific</td><td>BP220-212</td><td></td></tr><tr><td>100x protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td>Store at &amp;#8211; 20 C.</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-500</td><td></td></tr><tr><td>Trypan blue Stain 0.4%</td><td>GIBCO, by Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>10x Trypsin (0.5%)</td><td>GIBCO, by Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Trypsin Inhibitor </td><td>GIBCO, by Life Technologies</td><td>17075-029</td><td></td></tr></tbody></table>

probing for mitochondrial complex activity in human embryonic stem cells

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Mitochondria Have numerous roles in cell physiology from metabolism and energy production to program cell death to steroid hormone synthesis. Despite the importance of mitochondria in both fundamental cellular functions and disease, very little is known about the function of mitochondria in human embryonic stem cells and their differentiated progeny. In this video, we will show you how the mitochondria respiratory chain complexes of human amry stem cells can be analyzed using Ingel activity assays.Hi, I am Ivan OV from the laboratory of Michael Tetel in the Department of Pathology and Laboratory Medicine at the University of California Los Angeles. Today we will show you a procedure for assessing mitochondrial respiratory complex function in human embryonic stem cells. These in gel activity assess this procedure involves isolation of crude mitochondrial palate from human embryonic stem cells, separation of mitochondrial complexes of the exudative phosphorylation system with higher resolution clear native poly acrylamide gel electrophoresis, and subsequent in gel activity sa.So let's get started. The first step in this procedure is to isolate mitochondria from human embryonic stem cells. For this assay, we'll use human embryonic stem cells that have been grown on a matrigel covered plate in a feeder free condition.In order to collect mitochondria from our human amry stem cells, we start by washing them with warm one XPDS pH 7.4, and then trypsin the cells for five minutes. You can use one mil of one x trypsin per well in a six well plate. After five minutes, you can stop the reaction by adding an equal volume of fresh trypsin inhibitor one milligram per mil in one XPBS and harvest the cells.The collected cells are then centrifuge at 200 times G for five minutes at room temperature. After spinning, discard the supernatant and resus, suspend the pelleted cells in 0.8 to 1.7 milliliters of ice cold mitochondria isolation buffer with a freshly prepared cocktail of one x protease inhibitor. How much buffer you use depends on the size of the cell pellet you get After the pellet has been resuspended, incubate it on ice for about 10 minutes.Following the incubation on we down the cells with a homogenizer, this might take about 40 to 50 strokes. We can check for the complete list of homogenization by staining an aliquot of the DOD cells with trian blue and looking through a light microscope. It should look something like this.This is the first shot after 50 strokes with a downs. This is the second shot after 80 strokes with a downs. Once the cells are homogenized, centrifuge them at 20, 000 times G for 10 minutes at four degrees C.This will give us a crude mitochondrial pellet, which will also contain nuclei and larger cell fragments.Before collection of the pellet, we have to weigh the collection tube in order to estimate the subsequent pellet weight, pour out and discard the supernatant, and then weigh the precipitated crude mitochondrial pellet. By subtracting the weight of the tube at this point, one can proceed to solubilizing the pellet, which will be shown in the next step. In order to solubilize the crude mitochondrial pellet, we start by keeping the pellet on ice and vortexing with 35 microliters of ice cold solubilization buffer.For every 20 milligrams of pellet that we have detailed information on the buffer compounds is mentioned in the accompanying written protocol. Following vortexing and addition of solubilization buffer and before adding digit toin, we transfer the resuspended pellet into a tube appropriate for high speed centrifugation, and then we add 20 microliters of 10%weight per volume digit toin for 20 milligrams of pellet weight and mix by flicking the tube after it has been mixed thoroughly incubate the mitochondrial pellet for five to 10 minutes on ice. Doil beta D multiside or DDM or Triton X 100 can be substituted for digit toin to solubilized proteins.The choice of detergent and its quantity may affect the formation of supra complexes or multimeric forms of mitochondrial complexes following the incubation on ice. And once the pellet has been solubilized, we'll centrifuge it at a hundred thousand times G for 15 minutes at four degrees C, we will then collect the clear supernatant. We are now ready to prepare the supernatant for running on our gradient gel for native gradient gel electrophoresis of mitochondria.We recommend using an acrylamide gradient gel from five to 13%with a 3.5%stacking gel. To prepare the gel, we will use an acrylamide bis acrylamide mix as described in the written protocol. Acrylamide bis acrylamide solution is prepared in advance and stored at four degrees Celsius in a dark place.Acrylamide and bis acrylamide are toxic chemicals both as a powder and in liquid form, so special attention should be paid when you work with them. We combine the Acrylamide bis acrylamide solution with an aliquot of three x gel buffer composed of 1.5 molar six amino hexa noic acid, and 75 millimolar ole at pH 7.0. The amount of acrylamide bis acrylamide solution that we add to the buffer will depend on the percent gel that we are making to make the gradient gel.We will make one 4%mix and one 13%mix, and the stacking gel will be a 3.5%mix. Do not forget to add glycerol to the 13%mix. When all three mixtures are ready, we add an appropriate amount of ammonium per sulfate and temid to the mixtures and pour the mixtures into a gradient former casting chamber.The left part of the chamber contains the 5%mix. The right part of the chamber contains the 13%mix in a small magnetic stirring bar. We start to pour the gel by opening the right part of the chamber.Then immediately we open the left part of the chamber and let the 5%solution mix with a 13%solution to make the gradient. We add a small amount of isopropanol to the top of the gel to form a sharp straight border for the gel after polymerization. Approximately one hour later when the polymerization of the gradient gel is complete, we remove the isopropanol and pour the 3.5 stacking gel into the apparatus and insert a gel comb.We will assemble and run the gels in a cold room of about four to seven degrees Celsius. To run this gel, we'll be using two different buffers. In the anode chamber, we'll use a buffer containing 25 millimolar ole hydrochloric acid pH 7.0.The cathode buffer will contain 50 millimeter tric, 7.5 millimolar ole hydrochloric acid, 0.02%weight per volume, doil beta D multiside or DDM and 0.05%weight to volume, deoxy coate pH 7.0. In order to prepare the supernatant to be run in the gel that we've just poured, loading buffer is added to yield a final concentration of 5%weight to volume glycerol and 0.01%weight to volume. Ponto S, we typically add a 10 x stock solution containing glycerol and poncho s directly to the supernatants.Now we're ready to load the gel sample onto the gel. We will load the gel wells with a volume that contains 20 to 40 milligrams of the crude mitochondrial protein per lane. Immediately after loading, the gel is run at 100 volts.Once the sample has entered the stacking gel, the voltage can be raised to 500 volts with the current limited to 15 milliamps. Although we usually run the gel overnight at 70 to 100 volts, we stop the electrophoresis when a sharp line of the red zos dye approaches the gel front. Now that the gel has been run, we can analyze the activity of different protein complexes directly in the gel.The time of incubation for each assay depends on the efficiency of protein solubilization and the amount of material loaded per well. Complexes one, four and five usually yield stronger signals than complexes two and three. This is why staining time for complex two and three should be longer.To test the complex activity, we will need to use specific buffers for each complex. You can find the ingredients for the individual buffers in the written protocol. Now we will cut out individual lanes of the gel To perform the Ingel activity assays separately.For all the complexes, incubate the gel slice in their respective buffers for several hours at room temperature for all complexes except for complex five. After incubation, fix the gel in 50%methanol and 10%acetic acid for 15 to 45 minutes. The fixed gel is preserved in 10%acetic acid.You see here how the staining of complex one appears. There is one deep purple band in the gel slice that corresponds to complex one. Clear gels let you perform densitometry from mitochondrial complex activities and make comparative analyses.After dense cytometry, we usually dry the gel slices for long-term storage and or for scanning in order to obtain a high quality image complex, two stains the same purple color as one complex three and complex four. Both give an orange or yellowish staining band. Here are examples of complex two, three and four staining activities.We have just shown you how to analyze the activity of mitochondrial exudative ation complexes, which were extracted from human embryonic stem cells and separated by higher resolution, including native gel electros. So that's it. Thank you for watching and good luck with your experiment.

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